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Inhibitory effect of nordihydroguaiaretic acid on the frequency of micronuclei induced by methyl methanesulfonate in vivo
Mutation Research 441 Ž1999. 53–58
Inhibitory effect of nordihydroguaiaretic acid on the frequency of micronuclei induced by methyl methanesulfonate in vivo S. Dıaz ´ Barriga b, E. Madrigal-Bujaidar a
a,)
, P. Marquez ´
a
Laboratorio de Genetica, Escuela Nacional de Ciencias Biologicas, I.P.N. Carpio y Plan de Ayala, Sto. Tomas, C.P. 11340, ´ ´ Mexico D.F., Mexico b Laboratorio de Citogenetica ´ FES Cuautitlan, ´ UNAM, Mexico Received 3 May 1998; received in revised form 5 November 1998; accepted 10 February 1999
Abstract Nordihydroguaiaretic acid ŽNDGA. is an antioxidant originally obtained from plants of the genus Larrea. This chemical has shown antigenotoxic activity measuring gene mutations and sister-chromatid exchanges. The aim of this investigation was to determine if NDGA is also an antigenotoxic agent and can inhibit the induction of micronucleus ŽMN. formation by methyl methanesulfonate ŽMMS. in mouse. The frequency of micronucleated polychromatic erythrocytes ŽMPE. was scored for 4 days, and a MN induction curve by a singe injection of MMS Ž40 mgrkg. was obtained. The results of this experiment showed that the highest MN incidence was reached at the second day of exposure with a mean of 13.2% " 1.0. This value is more than 4 times the control mean. Thus, the modulatory study by NDGA was established at a 2-day exposure time using three doses Ž6.0, 11.0, and 17.0 mgrkg. against the damage induced by 40 mgrkg of MMS. The results of this study showed a significant reduction of the clastogenic damage at the two highest doses, where the inhibitory values corresponded to 62.2% and 66.7%, respectively. With respect to the ratio polychromatic erythrocytesrnormochromatic erythrocytes, a marked toxicity was detected with 2 days of MMS exposure; however, the combination of the two high doses of NDGA plus MMS significantly reduced the cytotoxic damage produced by MMS alone. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Micronucleus; Antigenotoxicity; Nordihydroguaiaretic acid; Methyl methanesulfonate
1. Introduction In a previous report we showed that nordihydroguaiaretic acid ŽNDGA. increased the rate of sister-chromatid exchanges ŽSCEs. in cultured human lymphocytes and in bone marrow cells of mice administered in vivo w1x. However, in another study ) Corresponding author. Fax: q52-5-3-96-35-03; E-mail: [email protected]
we also determined that non-toxic doses of this acid inhibited the production of SCEs by methyl methanesulfonate ŽMMS. in the two biological models mentioned w2x. This dual property of NDGA seems to be dose-dependent, and related to the specific mechanism of action. The detected modulatory activity of NDGA in mammalian cells is consistent with previous antimutagenic studies of this chemical, performed in the Ames–Salmonella typhimurium system with or with-
1383-5718r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 3 - 5 7 1 8 Ž 9 9 . 0 0 0 2 9 - 7
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S. Dıaz ´ Barriga et al.r Mutation Research 441 (1999) 53–58
out metabolic activation w3,4x. These studies analyzed the action of NDGA on the mutagenicity of chemicals such as methyl methanesulfonate, 2aminofluorene, aflatoxin B 1 and benzoŽ a.pyrene, each with a different mechanism of action. Beneficial effects of the compound have been established experimentally in cell culture systems and in different rodent models. These effects are related to neurodegenerative protection and significant reductions of bladder toxicity, neurotoxicity, and carcinogenicity w5–9x. The protective activity of NDGA may be due to its inhibitory action on several enzymes Že.g., the arachidonic acid cascade. or its blocking effect on oxidative damage w6,10–12x. Since NDGA appears to inhibit gene mutations and SCEs, the present investigation was designed to determine if NDGA can also reduce the frequency of micronuclei produced by MMS in peripheral blood of mice treated for 48 h.
toneally Ži.p.. to five animals. A blood sample from the tail of each mouse was obtained before the injection, and after 24, 48, 72, and 96 h of the initial administration. Two blood smears per animal were made at each time point, fixed for 3 min in methanol, and stained for 15 min in a Giemsa solution made in phosphate buffer at a pH of 6.8 w14x. One thousand polychromatic erythrocytes ŽPE. per mouse and per day were scored to determine the frequency of micronucleated cells. A statistical analysis was carried out according to the binomial comparison procedure described by Kastembaum and Bowman w15x. A polychromatic erythrocyte was identified by its larger size compared to a normochromatic erythrocyte as well as by its bluish color due to the cytoplasmic RNA remains. A micronucleus appeared as a round body with a well defined outline, and was also characterized by a purple color due to the presence of DNA. 2.3. Antimicronuclei induction by NDGA
2. Materials and methods 2.1. Chemicals and animals Nordihydroguaiaretic acid, MMS, and dimethyl sulfoxide ŽDMSO. were purchased from Sigma ŽSt Louis, MO.. The salts used to prepare the phosphate buffer Žmonobasic potassium phosphate, and dibasic sodium phosphate. were purchased from J.T. Baker ŽMexico City.. The Giemsa stain was obtained from Merck ŽMexico City., and mouse pellets were obtained from Purina ŽMexico City.. Five-week-old male mice with a mean weight of 25 g were obtained from the National Institute of Hygiene in Mexico City, from a strain originally developed by the National Institutes of Health, USA ŽNIH.. The animals were maintained at 238C in polypropylene cages and provided with standard feed and water ad libitum. 2.2. Micronuclei induction by MMS A preliminary study was conducted to establish a lethal dose-50 ŽLD50. for MMS following the method of Lorke w13x. The LD50 was found to be at 177.48 mgrkg, and 40 mgrkg of body weight was stated as the dose for genotoxicity studies. MMS was dissolved in distilled water and injected intraperi-
Based on the previous study we concluded that a 2-day time exposure to MMS was an appropriate period to significantly increase the rate of MN. Thus, the antigenotoxic study to test the ability of NDGA was designed to be scored at 2 days of exposure. The used doses of the chemical were selected based on its SCE and anti-SCE effects w1,2x. NDGA was dissolved in a 24% solution of DMSO Žmade in distilled water., and MMS in distilled water. The experimental protocol included five mice per group organized as follows: a control group treated with the solvent, three groups of mice administered with 6, 11, and 17 mgrkg of NDGA respectively, and three groups of animals administered with 6, 11, and 17 mgrkg of NDGA plus 40 mgrkg of MMS Žgroups 5,6, and 7.. All the mice were initially i.p. injected with the appropriate chemical Žthe DMSO" NDGA., and 30 min later the animals belonging to groups 5, 6, and 7 were i.p. injected with MMS. Two blood smears from the tail of each mouse were made after 48 h, fixed 3 min in methanol, and stained with Giemsa, pH 6.8 for 15 min. The frequency of micronucleated cells was determined in 1000 PE per animal, and the statistical significance was evaluated by using the method of Kastembaum and Bowman w15x.
S. Dıaz ´ Barriga et al.r Mutation Research 441 (1999) 53–58 Table 1 Frequency of micronucleated polychromatic erythrocytes ŽMPE. induced by 40 mgrkg of methyl methanesulfonate in mouse peripheral blood Sampling time day
MPE Žmean"S.E..
0 1 2 3 4
2.8"0.37 b 2.8"0.66 b 13.2"0.44 a 6.0"0.44 a,b 3.0"0.31b
a
Significant difference with respect to control value Žday 0.. Significant difference with respect to the second day value. Kastembaum–Bowman test, ps 0.05. b
We also determined the ratio of PE to normochromatic erythrocytes ŽNE. in 1000 cells per animal. The statistical significance of this parameter was evaluated with the Kruskall–Wallis and the Dunn’s Multiple Comparison tests w16,17x.
3. Results The ability of MMS to induce micronuclei ŽMN. is expressed in Table 1. We observed a significant increase in micronucleated polychromatic erythrocytes ŽMPE. at the second and third day of treatment with respect to the control value Žday 0.. On the second day, the maximum number of MN frequency
55
Ž) 4 times. was observed. This time point showed a statistically significant difference with respect to the MN frequencies obtained in the other days. The effect of NDGA to inhibit the MN induced by MMS is presented in Table 2. Concerning this endpoint, it was observed that before the treatment all experimental groups had a similar number of MPE with a mean variation ranging from 2.0 to 2.8 MPE, with no significant difference among them. The MPE mean for all groups before treatment was 2.4. At the end of the experiment, we found a similar response in the group of animals administered with the solvent as well as in those groups administered with the three tested doses of NDGA; the statistical analysis of these four experimental groups showed no significant differences among them. On the contrary, in mice administered with MMS alone, the frequency of MPE was significantly greater than that detected in the control animals. Nevertheless, an i.p. administration of NDGA in mice which were subsequently treated with MMS showed a protective effect on the genotoxic damage induced by the alkylating compound. The protection was observed with the three doses tested Ž6, 11, and 17 mgrkg of NDGA.; however, statistically significant results were obtained only with the two highest doses whose inhibitory values reached 62.2% and 66.7%, respectively. A linear regression analysis of data including the three antigenotoxic doses and the one obtained with MMS gave a correlation coefficient Ž r . of 0.85
Table 2 Effect of nordihydroguaiaretic acid ŽNDGA. on the frequency of micronucleated polychromatic erythrocytes ŽMPE. induced by methyl methanesulfonate ŽMMS. in mouse blood cells NDGA
Agent Žmgrkg.
MMS
Controla Ž6.0. Ž11.0. Ž17.0. Ž6.0. Ž11.0. Ž17.0. a
Ž40.0. Ž40.0. Ž40.0. Ž40.0.
MPEr1000 cells Ž0 h. Žmean " S.E..
MPEr1000 cells Ž48 h. Žmean " S.E..
Inhibition Ž%.
2.4 " 0.50 2.2 " 0.44 2.4 " 0.54 2.6 " 0.89 2.8 " 0.83 2.0 " 0.70 2.4 " 0.54 2.4 " 1.67
2.2 " 0.83 1.4 " 0.89 0.8 " 0.83 0.8 " 0.83 13.2 " 1.48 b 10.8 " 1.30 b 5.0 " 0.70 c 4.4 " 1.14 c
– – – – – 18.2 62.2 66.7
DMSO–distilled water Ž24% vrv.. Statistically significant difference with respect to control value. c Statistically significant difference with respect to MMS value, Kastenbaum–Bowman test, p s 0.05. b
S. Dıaz ´ Barriga et al.r Mutation Research 441 (1999) 53–58
56
Fig. 1. Regression line of the inhibitory activity of nordihydroguaiaretic acid ŽNDGA. on the micronuclei induced by methylmethanesulfonate in mice. Y s y0.568 X q 13, where X s dose of NDGA Žmgrkg. i.p. injected into mice Ž r s 0.8..
Ž Y s y0.568 X q 13.184. and suggested a dose-dependent response to NDGA with respect to its modulatory activity ŽFig. 1.. Our results show ŽTable 3. a decrease in PErNE ratio Ž76.4%. after 48 h of exposure to MMS when compared to the initial value; this indicates a marked bone marrow toxicity of MMS. A mild reduction of 19.6% in this ratio was also detected with the lower dose of NDGA and MMS. However, these effects were eliminated when we used the mutagen in com-
bination with each of the two high doses of NDGA Ž11 and 17 mgrkg.. 4. Discussion Methyl methanesulfonate is an alkylating agent known for its ability to react directly with DNA in vitro and in vivo. It produces genotoxic damage in different models. For example, it is known to induce mutations in the Ames–Salmonella typhimurium sys-
Table 3 Effect of nordihydroguaiaretic acid on methyl methanesulfonate in mouse blood cells. Frequency of polychromatic erythrocytes ŽPE. is with respect to the number of normochromatic erythrocytes ŽNE. NDGA
Agent Žmgrkg.
MMS
Controla Ž6.0. Ž11.0. Ž17.0. Ž6.0. Ž11.0. Ž17.0. a
Ž40.0. Ž40.0. Ž40.0. Ž40.0.
PErNE Žmean " S.D.. Ž0 h. b
PErNE Žmean " S.D.. Ž48 h. b
45.0 " 2.0 49.3 " 1.3 45.0 " 1.5 37.1 " 1.1 51.9 " 2.0 56.4 " 1.5 55.5 " 1.6 53.3 " 1.6
45.0 " 1.3 43.8 " 1.3 37.0 " 1.2 33.0 " 1.2 12.3 " 0.6 c 45.1 " 1.5 c 51.7 " 1.4 49.0 " 2.0
DMSO–distilled water Ž24% vrv.. Ratio obtained in 1000 cells per mouse. Five mice per dose. c Statistically significant difference with respect to the control value Ž0 h.. Kruskall–Wallis and Dunn’s Multiple Comparison tests, p s 0.05. b
S. Dıaz ´ Barriga et al.r Mutation Research 441 (1999) 53–58
tem, SCEs in cultured cells, increased levels of HGPRT-mutations in V79 Chinese hamster cells, chromosome breaking in several vegetables, and different types of DNA adducts w3,18–21x. Methyl methanesulfonate has been reported to induce dominant lethal mutations, heritable translocations and specific locus mutations w22x. Micronuclei induction by MMS has been studied in adult mice and fetal tissues with positive results w23,24x. In these studies the doses used vary from 10–80 mgrkg with clear significance at 50 mgrkg. Our study agrees with these data and confirms the potential of the chemical to induce chromosome fragments or abnormal segregation of whole chromosomes; thus, the observed MPE rate was significantly higher than the control rate. Furthermore, we found that a 2-day exposure after a single injection represented 4.7, 2.2, and 4.4 times the value obtained at day 1, 3, and 4 of MMS exposure. These results are congruent with the kinetics of the MN induction w25x. MacGregor et al. w25x established that after a single inoculation of chemicals, the maximum MN increase in mouse bone marrow cells was found after 1 day with a decrease of the MN value at the second day, where the maximum effect was detected in MN scored in peripheral blood cells. The genotoxic potential of NDGA in mammals has been studied w1x showing an increase of SCEs only with high levels of the compound. Based on this observation we selected doses for the present study which corresponded to the assumed non-genotoxic range.Our results confirmed this assumption with micronuclei, as its scoring showed that none of the NDGA tested doses induced a significant amount of MPE when administered alone. Our study also detected an efficient response of NDGA to decrease the MN induced by MMS, a fact that helps to understand the compound-modulatory action on different endpoints which presently include point mutations, SCEs and micronuclei. It has been suggested that NDGA acts as a blocking agent against specific chemicals by inhibiting the mixed function oxygenase system or by scavenging free radicals w6,10,12x.There is also evidence that it may interact directly with electrophile species, as its antimutagenic action may be exerted without S9 activation, and it inhibits the nitrosation products of methylurea and quench perylene free radicals effectively w3x.
57
These authors have suggested an interaction of NDGA with the methyl groups of MMS. In our study, the same doses of NDGA which produced an efficient antigenotoxic ability Ž11.0 and 17.0 mgrkg., also brought about a reduction on the cytotoxicity induced by MMS. This result agrees with published reports on other models w26,27x. NDGA blocked the cytotoxic action of several unsaturated fatty acids on HeLa cells and prevented the cytotoxicity of H 2 O 2 assessed by a colony formation assay in Chinese hamster V79 cells. Some phenolic compounds are known to suppress carcinogen-induced mutagenesis and neoplasia in experimental animals. An interesting example of the beneficial activities of this type of chemicals has been reported with respect to black and green tea w28,29x. Green tea in particular possesses several phenolic compounds and seems to have antipyretic, diuretic, antioxidative, radioprotective, antihepatotoxic and antimutagenic properties. NDGA is a polyphenolic lignan with a number of useful actions detected experimentally including the modulatory effect on MMS observed in the present study. This information may be relevant when we consider that experimental data on chemical mutagenesis suggest that mutagenic factors are important in carcinogenesis, and estimations based on epidemiological data indicate that about 75% of cancer cases are caused by environmental factors w30x. However, it is important to keep in mind the limitations generally observed in modulatory chemicals. In the case of NDGA, this chemical produces genotoxic damage at specific doses, which may be related to its conversion to semiquinone and quinone molecules and their subsequent interaction with the genetic material w1,31x. Furthermore, long-term studies in rats fed with the chemical Ž1 to 3%. showed histologic damage in the kidney, and a similar study with an extract of creosote bush in hamsters also showed disturbances in their testes and the accessory sex gland w32–34x. References w1x E. Madrigal-Bujaidar, S. Dıaz ´ Barriga, M. Cassani, D. Molina, G. Ponce, In vivo and in vitro induction of sisterchromatid exchanges by nordihydroguaiaretic acid, Mutation Res. 412 Ž1998. 139–144.
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S. Dıaz ´ Barriga et al.r Mutation Research 441 (1999) 53–58
w2x E. Madrigal-Bujaidar, S. Dıaz ´ Barriga, M. Cassani, P. Marquez, P. Revuelta, In vivo and in vitro antigenotoxic effect of nordihydroguaiaretic acid against SCEs induced by methyl methanesulfonate, Mutation Res. 419 Ž1998. 163–168. w3x Z.Y. Wang, R. Agarwal, Z.C. Zbou, D.R. Bickers, Antimutagenic and antitumorigenic activities of nordihydroguaiaretic acid, Mutation Res. 261 Ž1991. 155–162. w4x A.D. Rahimtula, P.K. Zachariah, P.J. O’Brien, The effects of antioxidants on the metabolism and mutagenicity of benzoŽ a.pyrene in vitro, Biochem. J. 164 Ž1977. 473–475. w5x L. Frasier, J.P. Kehrer, Effect of indomethacin, aspirin, nordihydroguaiaretic acid, and piperonyl butoxide on cyclophosphamide-induced bladder damage, Drug Chem. Toxicol. 16 Ž1993. 117–133. w6x Y. Goodman, M.R. Steiner, S.M. Steiner, M.P. Mattson, Nordihydroguaiaretic acid protects hippocampal neurons against amyloid B–peptide toxicity, and attenuates free radial and calcium accumulation, Brain Res. 654 Ž1994. 171–176. w7x S.M. Rothman, K.A. Yamada, N. Lancaster, Nordihydroguaiaretic acid attenuates MNDA neurotoxicity-action beyond the receptor, Neuropharmacology 32 Ž1993. 1279–1288. w8x D.L. McCormick, A.M. Spicer, Nordihydroguaiaretic acid suppression of rat mammary carcinogenesis induced by Nmethyl-N-nitrosourea, Cancer Lett. 37 Ž1987. 139–146. w9x A. Yu, T. Hashimura, Y. Nishio, H. Kanamaru, S. Fukuzawa, O.D. Yoshida, Anti-promoting effect of nordihydroguaiaretic acid on N-butyl-N-Ž4-hydroxybutyl. nitroso amine and sodium saccharin-induced rat urinary bladder carcinogenesis, Cancer Res. 83 Ž1992. 944–948. w10x R. Agarwal, Z.Y. Wang, D.P. BiK, H. Mukhtar, Nordihydroguaiaretic acid, an inhibitor of lipoxygenase, also inhibits cytochrome P-450 mediated monooxygenase activity in rat epidermal and hepatic microsomes, Drug Metabolism Disp. 19 Ž1991. 620–624. w11x J. Capdevilla, L. Gil, M. Orellana, L.J. Marnett, J.I. Mason, P. Yadagiri, J.R. Falck, Inhibitors of cytochrome P-450 dependent arachidonic acid metabolism, Arch. Biochem. Biophys. 261 Ž1988. 257–263. w12x L.R. Fitzpatrick, J.S. Bostwick, M. Renzetti, R.G. Pendleton, D.L. Decktor, Anti-inflammatory effects of various drugs on acetic acid induced colitis in the rat, Agents Actions 30 Ž1990. 393–402. w13x D. Lorke, A new approach to practical acute toxicity testing, Arch. Toxicol. 54 Ž1983. 275–287. w14x S. Dıaz E. ´ Barriga, E. Madrigal-Bujaidar, L. Ramırez, ´ Calderon, ´ B. Dıaz, ´ Genotoxic effect of capsaicin in a subchronic treatment in vivo, Mutation Res. 345 Ž1995. 105–109. w15x M.A. Kastenbaum, K.O. Bowman, Tables for determining the statistical significance of mutation frequencies, Mutation Res. 9 Ž1970. 527–549. w16x W.C. Schefler, Bioestadıstica, Fondo Educativo Interameri´ cano, Mexico, 1981, p. 226. ´ w17x J. Aceves, J. GraphPad InStat tm 931613Sq 1990–1993 GraphPad Software v2.03. w18x B. Lambert, M. Sten, D. Hellgren, D. Francesconi, Different SCE-inducing effects of HN2 and MMS in early and late G1 in human lymphocytes, Mutation Res. 251 Ž1984. 241–251. w19x R. Fiorio, R. Vellosi, G. Bronzetti, Effects of spermine on
w20x
w21x w22x
w23x
w24x
w25x
w26x
w27x
w28x
w29x
w30x
w31x
w32x
w33x
w34x
formation of HGPRT-mutants induced by ethylmethanesulfonate, methylmethanesulfonate, and mitomycin C in V79 Chinese hamster cells, Environ. Mol. Mutag. 23 Ž1994. 294–298. E.W. Vogel, M.J.M. Nivard, The subtlety of alkylating agents in reactions with biological macromolecules, Mutation Res. 305 Ž1994. 13–32. J. Moutschen, Mutagenesis with methyl methanesulfonate in mouse, Mutation Res. 8 Ž1969. 581–588. R.J. Cole, N. Taylor, J. Cole, C.F. Arlett, Short-term tests for transplacentally active carcinogens: I. Micronucleus formation in fetal and maternal mouse, Mutation Res. 80 Ž1981. 141–157. A.D. Tates, L. der Engelse, The role of short-lived lesions in the induction of micronuclei in rat liver by ethylnitrosourea and methyl methanesulphonate: the importance of experimental design, Mutation Res 210 Ž1989. 271–279. M. Shelby, K.R. Tindall, Mammalian germ cell mutagenicity of ENU, IPMS and MMS, chemicals selected for a transgenic mouse collaborative study, Mutation Res. 388 Ž1997. 99–109. J.T. MacGregor, C.M. Wehr, R.P. Henika, The in vivo erythrocyte micronucleus test: measurement at steady state increases assay efficiency and permits integration with toxicity studies, Fundam. Appl. Toxicol. 14 Ž1990. 513–522. P.S. Sagar, U.N. Das, R. Koratkar, G. Ramesh, M. Padma, G.S. Kumar, ŽHeLa. cells: relationship to free radicals and lipid peroxidation and its modulation by calmodulin antagonists, Cancer Lett. 63, 189–198. T. Nakayama, K. Hori, K. Terazawa, S. Kawakishi, Comparison of the cytotoxicity of different hydroperoxides in V79 cells, Free Radical Res. Commun. 14 Ž1991. 173–178. Z.Y. Wang, S.J. Cheng, Z.C. Zhou, M. Athar, M.W.A. Khan, D.R. Bickers, H. Mukhtar, Antimutagenic activity of green tea polyphenols, Mutation Res. 223 Ž1989. 273–285. J.H. Weisburger, Y. Hara, L. Dolan, F. Luo, B. Pittman, E. Zang, Tea polyphenols as inhibitors of mutagenicity of major classes of carcinogens, Mutation Res. 371 Ž1996. 57–63. C. Ramel, V.K. Alekperov, B.N. Ames, T. Kada, L.W. Wattenberg, Inhibitors of mutagenesis and their relevance to carcinogenesis, Mutation Res. 168 Ž1986. 47–65. M.J. van der Merwe, K. Jenkins, E. Theron, B.J. van der Walt, Interaction of the dicatechols rooperol and nordihydroguaiaretic acid with oxidative systems in the human blood. A structure–activity relationship, Biochem. Pharmacol. 45 Ž1993. 303–311. H.C. Grice, G. Becking, T. Goodman, Toxic properties of nordihydroguaiaretic acid, Fd. Cosmet. Toxicol. 6 Ž1968. 155–161. T. Goodman, D.V.M. Grice, G. Becking, F.A. Salem, A cystic nephropathy induced by nordihydroguaiaretic acid in the rat, Lab. Invest. 23 Ž1970. 93–107. H. Granados, R. Cardenas, Biliary calculi in the golden hamster: XXXVII. The prophylactic action of the creosote bush Ž Larrea tridentata. in pigmented cholelithiasis produced by vitamin A, Rev. Gastroenterol. Mex. 59 Ž1994. 31–35.
Inhibitory effect of nordihydroguaiaretic acid on the frequency of micronuclei induced by methyl methanesulfonate in vivo S. Dıaz ´ Barriga b, E. Madrigal-Bujaidar a
a,)
, P. Marquez ´
a
Laboratorio de Genetica, Escuela Nacional de Ciencias Biologicas, I.P.N. Carpio y Plan de Ayala, Sto. Tomas, C.P. 11340, ´ ´ Mexico D.F., Mexico b Laboratorio de Citogenetica ´ FES Cuautitlan, ´ UNAM, Mexico Received 3 May 1998; received in revised form 5 November 1998; accepted 10 February 1999
Abstract Nordihydroguaiaretic acid ŽNDGA. is an antioxidant originally obtained from plants of the genus Larrea. This chemical has shown antigenotoxic activity measuring gene mutations and sister-chromatid exchanges. The aim of this investigation was to determine if NDGA is also an antigenotoxic agent and can inhibit the induction of micronucleus ŽMN. formation by methyl methanesulfonate ŽMMS. in mouse. The frequency of micronucleated polychromatic erythrocytes ŽMPE. was scored for 4 days, and a MN induction curve by a singe injection of MMS Ž40 mgrkg. was obtained. The results of this experiment showed that the highest MN incidence was reached at the second day of exposure with a mean of 13.2% " 1.0. This value is more than 4 times the control mean. Thus, the modulatory study by NDGA was established at a 2-day exposure time using three doses Ž6.0, 11.0, and 17.0 mgrkg. against the damage induced by 40 mgrkg of MMS. The results of this study showed a significant reduction of the clastogenic damage at the two highest doses, where the inhibitory values corresponded to 62.2% and 66.7%, respectively. With respect to the ratio polychromatic erythrocytesrnormochromatic erythrocytes, a marked toxicity was detected with 2 days of MMS exposure; however, the combination of the two high doses of NDGA plus MMS significantly reduced the cytotoxic damage produced by MMS alone. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Micronucleus; Antigenotoxicity; Nordihydroguaiaretic acid; Methyl methanesulfonate
1. Introduction In a previous report we showed that nordihydroguaiaretic acid ŽNDGA. increased the rate of sister-chromatid exchanges ŽSCEs. in cultured human lymphocytes and in bone marrow cells of mice administered in vivo w1x. However, in another study ) Corresponding author. Fax: q52-5-3-96-35-03; E-mail: [email protected]
we also determined that non-toxic doses of this acid inhibited the production of SCEs by methyl methanesulfonate ŽMMS. in the two biological models mentioned w2x. This dual property of NDGA seems to be dose-dependent, and related to the specific mechanism of action. The detected modulatory activity of NDGA in mammalian cells is consistent with previous antimutagenic studies of this chemical, performed in the Ames–Salmonella typhimurium system with or with-
1383-5718r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 3 - 5 7 1 8 Ž 9 9 . 0 0 0 2 9 - 7
54
S. Dıaz ´ Barriga et al.r Mutation Research 441 (1999) 53–58
out metabolic activation w3,4x. These studies analyzed the action of NDGA on the mutagenicity of chemicals such as methyl methanesulfonate, 2aminofluorene, aflatoxin B 1 and benzoŽ a.pyrene, each with a different mechanism of action. Beneficial effects of the compound have been established experimentally in cell culture systems and in different rodent models. These effects are related to neurodegenerative protection and significant reductions of bladder toxicity, neurotoxicity, and carcinogenicity w5–9x. The protective activity of NDGA may be due to its inhibitory action on several enzymes Že.g., the arachidonic acid cascade. or its blocking effect on oxidative damage w6,10–12x. Since NDGA appears to inhibit gene mutations and SCEs, the present investigation was designed to determine if NDGA can also reduce the frequency of micronuclei produced by MMS in peripheral blood of mice treated for 48 h.
toneally Ži.p.. to five animals. A blood sample from the tail of each mouse was obtained before the injection, and after 24, 48, 72, and 96 h of the initial administration. Two blood smears per animal were made at each time point, fixed for 3 min in methanol, and stained for 15 min in a Giemsa solution made in phosphate buffer at a pH of 6.8 w14x. One thousand polychromatic erythrocytes ŽPE. per mouse and per day were scored to determine the frequency of micronucleated cells. A statistical analysis was carried out according to the binomial comparison procedure described by Kastembaum and Bowman w15x. A polychromatic erythrocyte was identified by its larger size compared to a normochromatic erythrocyte as well as by its bluish color due to the cytoplasmic RNA remains. A micronucleus appeared as a round body with a well defined outline, and was also characterized by a purple color due to the presence of DNA. 2.3. Antimicronuclei induction by NDGA
2. Materials and methods 2.1. Chemicals and animals Nordihydroguaiaretic acid, MMS, and dimethyl sulfoxide ŽDMSO. were purchased from Sigma ŽSt Louis, MO.. The salts used to prepare the phosphate buffer Žmonobasic potassium phosphate, and dibasic sodium phosphate. were purchased from J.T. Baker ŽMexico City.. The Giemsa stain was obtained from Merck ŽMexico City., and mouse pellets were obtained from Purina ŽMexico City.. Five-week-old male mice with a mean weight of 25 g were obtained from the National Institute of Hygiene in Mexico City, from a strain originally developed by the National Institutes of Health, USA ŽNIH.. The animals were maintained at 238C in polypropylene cages and provided with standard feed and water ad libitum. 2.2. Micronuclei induction by MMS A preliminary study was conducted to establish a lethal dose-50 ŽLD50. for MMS following the method of Lorke w13x. The LD50 was found to be at 177.48 mgrkg, and 40 mgrkg of body weight was stated as the dose for genotoxicity studies. MMS was dissolved in distilled water and injected intraperi-
Based on the previous study we concluded that a 2-day time exposure to MMS was an appropriate period to significantly increase the rate of MN. Thus, the antigenotoxic study to test the ability of NDGA was designed to be scored at 2 days of exposure. The used doses of the chemical were selected based on its SCE and anti-SCE effects w1,2x. NDGA was dissolved in a 24% solution of DMSO Žmade in distilled water., and MMS in distilled water. The experimental protocol included five mice per group organized as follows: a control group treated with the solvent, three groups of mice administered with 6, 11, and 17 mgrkg of NDGA respectively, and three groups of animals administered with 6, 11, and 17 mgrkg of NDGA plus 40 mgrkg of MMS Žgroups 5,6, and 7.. All the mice were initially i.p. injected with the appropriate chemical Žthe DMSO" NDGA., and 30 min later the animals belonging to groups 5, 6, and 7 were i.p. injected with MMS. Two blood smears from the tail of each mouse were made after 48 h, fixed 3 min in methanol, and stained with Giemsa, pH 6.8 for 15 min. The frequency of micronucleated cells was determined in 1000 PE per animal, and the statistical significance was evaluated by using the method of Kastembaum and Bowman w15x.
S. Dıaz ´ Barriga et al.r Mutation Research 441 (1999) 53–58 Table 1 Frequency of micronucleated polychromatic erythrocytes ŽMPE. induced by 40 mgrkg of methyl methanesulfonate in mouse peripheral blood Sampling time day
MPE Žmean"S.E..
0 1 2 3 4
2.8"0.37 b 2.8"0.66 b 13.2"0.44 a 6.0"0.44 a,b 3.0"0.31b
a
Significant difference with respect to control value Žday 0.. Significant difference with respect to the second day value. Kastembaum–Bowman test, ps 0.05. b
We also determined the ratio of PE to normochromatic erythrocytes ŽNE. in 1000 cells per animal. The statistical significance of this parameter was evaluated with the Kruskall–Wallis and the Dunn’s Multiple Comparison tests w16,17x.
3. Results The ability of MMS to induce micronuclei ŽMN. is expressed in Table 1. We observed a significant increase in micronucleated polychromatic erythrocytes ŽMPE. at the second and third day of treatment with respect to the control value Žday 0.. On the second day, the maximum number of MN frequency
55
Ž) 4 times. was observed. This time point showed a statistically significant difference with respect to the MN frequencies obtained in the other days. The effect of NDGA to inhibit the MN induced by MMS is presented in Table 2. Concerning this endpoint, it was observed that before the treatment all experimental groups had a similar number of MPE with a mean variation ranging from 2.0 to 2.8 MPE, with no significant difference among them. The MPE mean for all groups before treatment was 2.4. At the end of the experiment, we found a similar response in the group of animals administered with the solvent as well as in those groups administered with the three tested doses of NDGA; the statistical analysis of these four experimental groups showed no significant differences among them. On the contrary, in mice administered with MMS alone, the frequency of MPE was significantly greater than that detected in the control animals. Nevertheless, an i.p. administration of NDGA in mice which were subsequently treated with MMS showed a protective effect on the genotoxic damage induced by the alkylating compound. The protection was observed with the three doses tested Ž6, 11, and 17 mgrkg of NDGA.; however, statistically significant results were obtained only with the two highest doses whose inhibitory values reached 62.2% and 66.7%, respectively. A linear regression analysis of data including the three antigenotoxic doses and the one obtained with MMS gave a correlation coefficient Ž r . of 0.85
Table 2 Effect of nordihydroguaiaretic acid ŽNDGA. on the frequency of micronucleated polychromatic erythrocytes ŽMPE. induced by methyl methanesulfonate ŽMMS. in mouse blood cells NDGA
Agent Žmgrkg.
MMS
Controla Ž6.0. Ž11.0. Ž17.0. Ž6.0. Ž11.0. Ž17.0. a
Ž40.0. Ž40.0. Ž40.0. Ž40.0.
MPEr1000 cells Ž0 h. Žmean " S.E..
MPEr1000 cells Ž48 h. Žmean " S.E..
Inhibition Ž%.
2.4 " 0.50 2.2 " 0.44 2.4 " 0.54 2.6 " 0.89 2.8 " 0.83 2.0 " 0.70 2.4 " 0.54 2.4 " 1.67
2.2 " 0.83 1.4 " 0.89 0.8 " 0.83 0.8 " 0.83 13.2 " 1.48 b 10.8 " 1.30 b 5.0 " 0.70 c 4.4 " 1.14 c
– – – – – 18.2 62.2 66.7
DMSO–distilled water Ž24% vrv.. Statistically significant difference with respect to control value. c Statistically significant difference with respect to MMS value, Kastenbaum–Bowman test, p s 0.05. b
S. Dıaz ´ Barriga et al.r Mutation Research 441 (1999) 53–58
56
Fig. 1. Regression line of the inhibitory activity of nordihydroguaiaretic acid ŽNDGA. on the micronuclei induced by methylmethanesulfonate in mice. Y s y0.568 X q 13, where X s dose of NDGA Žmgrkg. i.p. injected into mice Ž r s 0.8..
Ž Y s y0.568 X q 13.184. and suggested a dose-dependent response to NDGA with respect to its modulatory activity ŽFig. 1.. Our results show ŽTable 3. a decrease in PErNE ratio Ž76.4%. after 48 h of exposure to MMS when compared to the initial value; this indicates a marked bone marrow toxicity of MMS. A mild reduction of 19.6% in this ratio was also detected with the lower dose of NDGA and MMS. However, these effects were eliminated when we used the mutagen in com-
bination with each of the two high doses of NDGA Ž11 and 17 mgrkg.. 4. Discussion Methyl methanesulfonate is an alkylating agent known for its ability to react directly with DNA in vitro and in vivo. It produces genotoxic damage in different models. For example, it is known to induce mutations in the Ames–Salmonella typhimurium sys-
Table 3 Effect of nordihydroguaiaretic acid on methyl methanesulfonate in mouse blood cells. Frequency of polychromatic erythrocytes ŽPE. is with respect to the number of normochromatic erythrocytes ŽNE. NDGA
Agent Žmgrkg.
MMS
Controla Ž6.0. Ž11.0. Ž17.0. Ž6.0. Ž11.0. Ž17.0. a
Ž40.0. Ž40.0. Ž40.0. Ž40.0.
PErNE Žmean " S.D.. Ž0 h. b
PErNE Žmean " S.D.. Ž48 h. b
45.0 " 2.0 49.3 " 1.3 45.0 " 1.5 37.1 " 1.1 51.9 " 2.0 56.4 " 1.5 55.5 " 1.6 53.3 " 1.6
45.0 " 1.3 43.8 " 1.3 37.0 " 1.2 33.0 " 1.2 12.3 " 0.6 c 45.1 " 1.5 c 51.7 " 1.4 49.0 " 2.0
DMSO–distilled water Ž24% vrv.. Ratio obtained in 1000 cells per mouse. Five mice per dose. c Statistically significant difference with respect to the control value Ž0 h.. Kruskall–Wallis and Dunn’s Multiple Comparison tests, p s 0.05. b
S. Dıaz ´ Barriga et al.r Mutation Research 441 (1999) 53–58
tem, SCEs in cultured cells, increased levels of HGPRT-mutations in V79 Chinese hamster cells, chromosome breaking in several vegetables, and different types of DNA adducts w3,18–21x. Methyl methanesulfonate has been reported to induce dominant lethal mutations, heritable translocations and specific locus mutations w22x. Micronuclei induction by MMS has been studied in adult mice and fetal tissues with positive results w23,24x. In these studies the doses used vary from 10–80 mgrkg with clear significance at 50 mgrkg. Our study agrees with these data and confirms the potential of the chemical to induce chromosome fragments or abnormal segregation of whole chromosomes; thus, the observed MPE rate was significantly higher than the control rate. Furthermore, we found that a 2-day exposure after a single injection represented 4.7, 2.2, and 4.4 times the value obtained at day 1, 3, and 4 of MMS exposure. These results are congruent with the kinetics of the MN induction w25x. MacGregor et al. w25x established that after a single inoculation of chemicals, the maximum MN increase in mouse bone marrow cells was found after 1 day with a decrease of the MN value at the second day, where the maximum effect was detected in MN scored in peripheral blood cells. The genotoxic potential of NDGA in mammals has been studied w1x showing an increase of SCEs only with high levels of the compound. Based on this observation we selected doses for the present study which corresponded to the assumed non-genotoxic range.Our results confirmed this assumption with micronuclei, as its scoring showed that none of the NDGA tested doses induced a significant amount of MPE when administered alone. Our study also detected an efficient response of NDGA to decrease the MN induced by MMS, a fact that helps to understand the compound-modulatory action on different endpoints which presently include point mutations, SCEs and micronuclei. It has been suggested that NDGA acts as a blocking agent against specific chemicals by inhibiting the mixed function oxygenase system or by scavenging free radicals w6,10,12x.There is also evidence that it may interact directly with electrophile species, as its antimutagenic action may be exerted without S9 activation, and it inhibits the nitrosation products of methylurea and quench perylene free radicals effectively w3x.
57
These authors have suggested an interaction of NDGA with the methyl groups of MMS. In our study, the same doses of NDGA which produced an efficient antigenotoxic ability Ž11.0 and 17.0 mgrkg., also brought about a reduction on the cytotoxicity induced by MMS. This result agrees with published reports on other models w26,27x. NDGA blocked the cytotoxic action of several unsaturated fatty acids on HeLa cells and prevented the cytotoxicity of H 2 O 2 assessed by a colony formation assay in Chinese hamster V79 cells. Some phenolic compounds are known to suppress carcinogen-induced mutagenesis and neoplasia in experimental animals. An interesting example of the beneficial activities of this type of chemicals has been reported with respect to black and green tea w28,29x. Green tea in particular possesses several phenolic compounds and seems to have antipyretic, diuretic, antioxidative, radioprotective, antihepatotoxic and antimutagenic properties. NDGA is a polyphenolic lignan with a number of useful actions detected experimentally including the modulatory effect on MMS observed in the present study. This information may be relevant when we consider that experimental data on chemical mutagenesis suggest that mutagenic factors are important in carcinogenesis, and estimations based on epidemiological data indicate that about 75% of cancer cases are caused by environmental factors w30x. However, it is important to keep in mind the limitations generally observed in modulatory chemicals. In the case of NDGA, this chemical produces genotoxic damage at specific doses, which may be related to its conversion to semiquinone and quinone molecules and their subsequent interaction with the genetic material w1,31x. Furthermore, long-term studies in rats fed with the chemical Ž1 to 3%. showed histologic damage in the kidney, and a similar study with an extract of creosote bush in hamsters also showed disturbances in their testes and the accessory sex gland w32–34x. References w1x E. Madrigal-Bujaidar, S. Dıaz ´ Barriga, M. Cassani, D. Molina, G. Ponce, In vivo and in vitro induction of sisterchromatid exchanges by nordihydroguaiaretic acid, Mutation Res. 412 Ž1998. 139–144.
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w2x E. Madrigal-Bujaidar, S. Dıaz ´ Barriga, M. Cassani, P. Marquez, P. Revuelta, In vivo and in vitro antigenotoxic effect of nordihydroguaiaretic acid against SCEs induced by methyl methanesulfonate, Mutation Res. 419 Ž1998. 163–168. w3x Z.Y. Wang, R. Agarwal, Z.C. Zbou, D.R. Bickers, Antimutagenic and antitumorigenic activities of nordihydroguaiaretic acid, Mutation Res. 261 Ž1991. 155–162. w4x A.D. Rahimtula, P.K. Zachariah, P.J. O’Brien, The effects of antioxidants on the metabolism and mutagenicity of benzoŽ a.pyrene in vitro, Biochem. J. 164 Ž1977. 473–475. w5x L. Frasier, J.P. Kehrer, Effect of indomethacin, aspirin, nordihydroguaiaretic acid, and piperonyl butoxide on cyclophosphamide-induced bladder damage, Drug Chem. Toxicol. 16 Ž1993. 117–133. w6x Y. Goodman, M.R. Steiner, S.M. Steiner, M.P. Mattson, Nordihydroguaiaretic acid protects hippocampal neurons against amyloid B–peptide toxicity, and attenuates free radial and calcium accumulation, Brain Res. 654 Ž1994. 171–176. w7x S.M. Rothman, K.A. Yamada, N. Lancaster, Nordihydroguaiaretic acid attenuates MNDA neurotoxicity-action beyond the receptor, Neuropharmacology 32 Ž1993. 1279–1288. w8x D.L. McCormick, A.M. Spicer, Nordihydroguaiaretic acid suppression of rat mammary carcinogenesis induced by Nmethyl-N-nitrosourea, Cancer Lett. 37 Ž1987. 139–146. w9x A. Yu, T. Hashimura, Y. Nishio, H. Kanamaru, S. Fukuzawa, O.D. Yoshida, Anti-promoting effect of nordihydroguaiaretic acid on N-butyl-N-Ž4-hydroxybutyl. nitroso amine and sodium saccharin-induced rat urinary bladder carcinogenesis, Cancer Res. 83 Ž1992. 944–948. w10x R. Agarwal, Z.Y. Wang, D.P. BiK, H. Mukhtar, Nordihydroguaiaretic acid, an inhibitor of lipoxygenase, also inhibits cytochrome P-450 mediated monooxygenase activity in rat epidermal and hepatic microsomes, Drug Metabolism Disp. 19 Ž1991. 620–624. w11x J. Capdevilla, L. Gil, M. Orellana, L.J. Marnett, J.I. Mason, P. Yadagiri, J.R. Falck, Inhibitors of cytochrome P-450 dependent arachidonic acid metabolism, Arch. Biochem. Biophys. 261 Ž1988. 257–263. w12x L.R. Fitzpatrick, J.S. Bostwick, M. Renzetti, R.G. Pendleton, D.L. Decktor, Anti-inflammatory effects of various drugs on acetic acid induced colitis in the rat, Agents Actions 30 Ž1990. 393–402. w13x D. Lorke, A new approach to practical acute toxicity testing, Arch. Toxicol. 54 Ž1983. 275–287. w14x S. Dıaz E. ´ Barriga, E. Madrigal-Bujaidar, L. Ramırez, ´ Calderon, ´ B. Dıaz, ´ Genotoxic effect of capsaicin in a subchronic treatment in vivo, Mutation Res. 345 Ž1995. 105–109. w15x M.A. Kastenbaum, K.O. Bowman, Tables for determining the statistical significance of mutation frequencies, Mutation Res. 9 Ž1970. 527–549. w16x W.C. Schefler, Bioestadıstica, Fondo Educativo Interameri´ cano, Mexico, 1981, p. 226. ´ w17x J. Aceves, J. GraphPad InStat tm 931613Sq 1990–1993 GraphPad Software v2.03. w18x B. Lambert, M. Sten, D. Hellgren, D. Francesconi, Different SCE-inducing effects of HN2 and MMS in early and late G1 in human lymphocytes, Mutation Res. 251 Ž1984. 241–251. w19x R. Fiorio, R. Vellosi, G. Bronzetti, Effects of spermine on
w20x
w21x w22x
w23x
w24x
w25x
w26x
w27x
w28x
w29x
w30x
w31x
w32x
w33x
w34x
formation of HGPRT-mutants induced by ethylmethanesulfonate, methylmethanesulfonate, and mitomycin C in V79 Chinese hamster cells, Environ. Mol. Mutag. 23 Ž1994. 294–298. E.W. Vogel, M.J.M. Nivard, The subtlety of alkylating agents in reactions with biological macromolecules, Mutation Res. 305 Ž1994. 13–32. J. Moutschen, Mutagenesis with methyl methanesulfonate in mouse, Mutation Res. 8 Ž1969. 581–588. R.J. Cole, N. Taylor, J. Cole, C.F. Arlett, Short-term tests for transplacentally active carcinogens: I. Micronucleus formation in fetal and maternal mouse, Mutation Res. 80 Ž1981. 141–157. A.D. Tates, L. der Engelse, The role of short-lived lesions in the induction of micronuclei in rat liver by ethylnitrosourea and methyl methanesulphonate: the importance of experimental design, Mutation Res 210 Ž1989. 271–279. M. Shelby, K.R. Tindall, Mammalian germ cell mutagenicity of ENU, IPMS and MMS, chemicals selected for a transgenic mouse collaborative study, Mutation Res. 388 Ž1997. 99–109. J.T. MacGregor, C.M. Wehr, R.P. Henika, The in vivo erythrocyte micronucleus test: measurement at steady state increases assay efficiency and permits integration with toxicity studies, Fundam. Appl. Toxicol. 14 Ž1990. 513–522. P.S. Sagar, U.N. Das, R. Koratkar, G. Ramesh, M. Padma, G.S. Kumar, ŽHeLa. cells: relationship to free radicals and lipid peroxidation and its modulation by calmodulin antagonists, Cancer Lett. 63, 189–198. T. Nakayama, K. Hori, K. Terazawa, S. Kawakishi, Comparison of the cytotoxicity of different hydroperoxides in V79 cells, Free Radical Res. Commun. 14 Ž1991. 173–178. Z.Y. Wang, S.J. Cheng, Z.C. Zhou, M. Athar, M.W.A. Khan, D.R. Bickers, H. Mukhtar, Antimutagenic activity of green tea polyphenols, Mutation Res. 223 Ž1989. 273–285. J.H. Weisburger, Y. Hara, L. Dolan, F. Luo, B. Pittman, E. Zang, Tea polyphenols as inhibitors of mutagenicity of major classes of carcinogens, Mutation Res. 371 Ž1996. 57–63. C. Ramel, V.K. Alekperov, B.N. Ames, T. Kada, L.W. Wattenberg, Inhibitors of mutagenesis and their relevance to carcinogenesis, Mutation Res. 168 Ž1986. 47–65. M.J. van der Merwe, K. Jenkins, E. Theron, B.J. van der Walt, Interaction of the dicatechols rooperol and nordihydroguaiaretic acid with oxidative systems in the human blood. A structure–activity relationship, Biochem. Pharmacol. 45 Ž1993. 303–311. H.C. Grice, G. Becking, T. Goodman, Toxic properties of nordihydroguaiaretic acid, Fd. Cosmet. Toxicol. 6 Ž1968. 155–161. T. Goodman, D.V.M. Grice, G. Becking, F.A. Salem, A cystic nephropathy induced by nordihydroguaiaretic acid in the rat, Lab. Invest. 23 Ž1970. 93–107. H. Granados, R. Cardenas, Biliary calculi in the golden hamster: XXXVII. The prophylactic action of the creosote bush Ž Larrea tridentata. in pigmented cholelithiasis produced by vitamin A, Rev. Gastroenterol. Mex. 59 Ž1994. 31–35.